An emerging approach to treat patients with heart failure from non-ischemic cardiomyopathy (NICM) involves delivery of mesenchymal stem cells (MSCs) that can that can improve CM performance in cell culture and in animal models, and are being tested in ongoing clinical trials. The functional benefits of MSC-therapy may involve a variety of mechanisms, but it remains unclear whether transplanted cells act primarily through direct cell-cell interactions, or through indirect paracrine signaling via soluble factors or via special microvesicles called exosomes that can transfer molecular cargo. Understanding MSC-enhanced CM function could lead to improved cardiotherapeutics, but progress has been hampered by the limits of existing models systems for understanding paracrine signaling in the cardiac niche environment. Directly addressing an NHLBI topic of special interest (HL-142) on the role of exosomes as paracrine signal mediators in cardiovascular disease, this proposal aims to use 3D human engineered cardiac tissue (hECT) as a controllable biomimetic in vitro model of native human myocardium in order to identify the primary factors underlying MSC-mediated effects on cardiomyocyte contractile function. A novel multi-hECT bioreactor system with integrated force-sensing technology has generated preliminary data supporting a predominant effect of extrinsic paracrine signaling mechanisms, including bioactive secreted exosomes, that significantly exceed the benefits of direct coupling between MSCs and hCMs in human engineered cardiac tissues. The governing hypothesis is that MSC treatment causes direct enhancement of cardiomyocyte contractile function primarily through paracrine signaling mechanisms involving secreted exosomes that can be identified, isolated, deconstructed and delivered as an alternative therapy for non-ischemic heart failure. Specific Aim 1 will resolve the environmental conditions that maximize MSC paracrine enhancement of hECT contractile performance, advancing our understanding of specific biophysical stimuli that modulate cardioactive signaling processes. Aim 2 will identify the role of exosomes and their molecular cargo in MSC-mediated contractile enhancement of hECTs by evaluating the potency of hMSC exosomes and cargo on hECT contractile function (Sub-aim 2a) and determining the molecular identity of lead inotropic compounds from hMSC exosome cargo (Sub-aim 2b). Finally, Aim 3 will evaluate the therapeutic efficacy of delivered hMSC exosome-derived cardiotropic factors on recovery of contractility using in vitro (Sub-aim 3a) and in vivo (Sub-aim 3b) models of non-ischemic heart failure. By capturing the benefits of MSC therapy while circumventing the potential risks of live cell implantation, this proposal may lead to improved treatment options for patients who suffer heart failure from non-ischemic cardiomyopathy.